Overall PROJECT SUMMARY Defects in DNA repair machines are at the heart of what leads to cancer-causing mutations, what prevents cancer, what interferes with cancer treatment, and what is the Achilles? heel for cancer-targeted treatment. For NCI, SBDR coordinates leaders in DNA repair (DR) to work together synergistically to provide a comprehensive, mechanistic understanding of DR processes. SBDR removes bottlenecks within individual laboratories and promotes the concerted efforts of multidisciplinary researchers with expertise in different facets of DR. The SBDR Projects and Cores together enable a comprehensive cross-pathway knowledge of dynamic multi-functional DR machines ? an understanding that can only be achieved through multi-disciplinary approaches and concerted efforts by multiple groups. The goals of SBDR are 1) to develop structure-based, mechanistic foundation for an actionable understanding of dynamic, multi-functional DR molecular complexes suitable for cancer biology, prognosis, and predispositions, and 2) to enable an integrated quantitative and mechanistic knowledge of DR machines, pathways, and intersections with replication and apoptosis sufficient to aid prediction and intervention for cancer biology by bridging the gaps from mutant sequences to system level correlation. By integrating analyses of major DR and damage response pathways with replication, SBDR will provide detailed and comprehensive information on the maintenance of genetic integrity spanning from specific proteins and complexes to pathways, networks, and signaling. We will apply knowledge-based determination and integration of structural, biochemical, and biological data on DR protein interactions, modifications, and complexes acting in the six Projects. Our multifaceted structure-based strategy both dissects multiple activities of multi-functional complexes and defines how these proteins act in multiple pathways. In concert, SBDR Projects and Cores will accomplish 4 Program Aims: 1) Determine definitive and biologically validated structures of DR complexes, interfaces, & conformations; 2) Dissect the multi-functionality of DR machineries tested by structurally-based examination of separation-of-function mutations and chemical inhibitors; 3) Define crosstalk for DR pathway interactions and delineate and test how DR pathway choice is made; and 4) Discover and characterize synthetic lethalities brought about by either mutations or chemical agents that target a specific DR activity that is lethal only in the context of perturbation of another DR defect to control biological outcomes to DNA damage. SBDR will inform cancer biology by providing a comprehensive mechanistic knowledge of how cells respond to DNA damage and how multi-functional DR proteins act in the context of other DNA processes. SBDR and the new Project 6 on DNA and RNA alkylation response will advance understanding of how DR mutations may differentially impact cancer susceptibility and patient risk. SBDR will aid research employing Cancer Genome Atlas mutations and system level correlations as well as consequent therapeutic strategies by providing mechanistic and predictive insights.